High lysine maize compositions and event LY038 maize plants

ABSTRACT

Disclosed herein are novel DNA constructs encoding dihydrodipicolinic acid synthase that are useful for generating transgenic events. Also provided are transgenic plants harboring such DNA constructs, the expression of which results in increased lysine in the plant or plant product. Also provided are maize plants having LY038 Event and progeny thereof having this event. Additionally disclosed are assays for detecting the presence of a lysine-increasing transgenic event based on the DNA sequence of the exogenous DNA construct inserted into the maize genome and of genomic sequences flanking the insertion site.

This application claims benefit under 35 USC § 119(e) of U.S.provisional application Ser. No. 60/529,182 filed 11 Dec. 2003, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology.More specifically, the present invention relates to transgenic maizewith increased lysine, and to assays and methods for identifying thespecific exogenous DNA providing increased lysine.

BACKGROUND OF THE INVENTION

Zea mays, commonly known as maize and corn, is a grain widely used inanimal feed. The grain, i.e., kernel, is a source of protein, starch,and oil for swine, cattle, and poultry. Of the ten amino acids deemedessential in a mixed grain feed source, corn is particularly limiting inlysine, threonine, and methionine. The lack of these amino acids,especially lysine, requires that feed corn or corn meal be supplementedwith these nutrients, often provided by the addition of soybean meal. Itwould be of benefit to the art to increase the level of lysine in cornkernel as a means of making the seed and meal more nutritious as ananimal feed.

In order to increase levels of lysine using a molecular biologicalapproach, a feedback-insensitive version of at least one of the lysinepathway enzymes, namely dihydrodipicolinic acid synthase (referred toherein as DHDPS), has been identified and employed. A bacterial DHDPSgene isolated from E. coli has been shown in vitro to be about 200-foldless sensitive to inhibition by increases in lysine levels and, whenintroduced into transgenic tobacco, over-expression of the E. coli DHDPSgene resulted in increased levels of lysine in leaf tissue (Glassman etal., U.S. Pat. No. 5,288,300). Falco et al. disclose transgenic plantswith increased levels of lysine in the seed and genes useful for theproduction of such transgenic plants (U.S. Pat. Nos. 5,773,691 and6,459,019; U.S. Patent Application Publication 2003/0056242, each ofwhich is incorporated herein by reference in its entirety). In thesereports, Falco et al. describe the isolation and use offeedback-insensitive DHDPS from E. coli and DHDPS from Corynebacterium(also known as cordapA) to generate transgenic rapeseed, tobacco, maize,and soybean plants with increased levels of lysine in the seed. Formaize, Falco et al. report an approximately 130% increase in free lysinein kernels transformed with the cordapA gene relative to non-transformedkernels.

It would be advantageous to be able to detect the presence or absence ofa particular transgene in a plant or seed, or progeny of such plants orseeds, not only with respect to the transgene itself, but also withrespect to its location in the genome of a host plant or seed.Identification with respect to location further provides identificationof the transgenic event by which a genetic engineer inserted thetransgene into the progenitor plant of the plant or seed.

SUMMARY OF THE INVENTION

The present invention provides constructs useful for generatingtransgenic events and materials and methods useful for identifyingparticular transgenic events that resulted in transgenic plants thataccumulate higher levels of lysine than do closely related plants thatdo not include the construct. In particular, this invention comprises aline of marker-free transgenic maize comprising a specific exogenous DNAthat was introduced via standard maize transformation, referred toherein as “the LY038 Event” or “Event LY038.” The present inventionfurther provides a method for detecting the presence or absence of theLY038 Event in DNA obtained from maize plants, seeds, or tissue samples.The maize plant of the present invention comprising Event LY038 exhibitsincreased lysine in the kernel relative to a progenitor or othersubstantially related plant. In addition, the present invention providesan exogenous DNA construct comprising a maize globulin 1 promoter, arice actin 1 intron, a maize dihydrodipicolinate synthase chloroplasttransit peptide-encoding DNA molecule, a Corynebacteriumdihydrodipicolinate synthase-encoding DNA molecule, and a maize globulin1 3′ untranslated region that, when operably linked and expressed intransgenic plant cells and plants, results in increased lysine contentin the kernel or parts thereof or a processed product derived from thekernel or plant. In another embodiment, the construct further comprisesa lox site, present as a component of a mechanism for removing themarker gene used to identify successful transformants.

With respect to identifying a plant or seed derived from a particulartransgenic event, compositions and methods are provided for detectingthe presence of the genomic insertion region from a novel maize plantcomprising Event LY038, i.e., the site in the genome where the constructresides. DNA molecules are provided that comprise at least a portion ofthe exogenous DNA inserted into the genome and a portion of the DNA fromthe maize genome flanking the insertion site (referred to herein as a“junction sequence”).

In yet another embodiment of the present invention, novel DNA moleculesare provided that comprise at least about 20 base pairs of SEQ ID NO: 1or 2, where base pairs 1781–1782 or 200–201, respectively, are included.Further provided by the present invention are DNA molecules comprisingthe sequence of an amplicon having the sequence of SEQ ID NO: 6 obtainedby a DNA amplification method using primers of SEQ ID NOs: 3 and 4; anda hybridization probe complementary to said amplicon, having thesequence of SEQ ID NO: 5 and complements thereof. DNA molecules havingthe sequence of SEQ ID NO: 5 or 6 span the junction between theexogenous DNA and flanking maize genomic DNA and are diagnostic forEvent LY038 DNA when used in suitable analytical tests. Other preferredDNA molecules of the present invention that span the junction of theexogenous DNA/genomic insertion region of Event LY038 are moleculeshaving the sequence of SEQ ID NOs: 1, 2, and 11, and complementsthereof. A stably transformed maize plant or seed comprising thesemolecules is another aspect of this invention.

Primers are said to be of “sufficient length” when they are of a lengththat allows the primer to function in a PCR reaction and specificallyamplify a target sequence; a length of about 11 nucleotides or more issufficient, more preferably about 18 nucleotides or more, yet morepreferably about 24 nucleotides or more, even more preferably about 30nucleotides is sufficient to perform and specifically amplify a targetsequence. One skilled in the art would know that a primer of evengreater length than about 30 nucleotides can be usefully employed in aPCR reaction and, accordingly, is of a sufficient length.

PCR primers useful for identifying Event LY038 comprise a sufficientlength of a transgene portion of the DNA sequence of SEQ ID NO: 1 and asufficient length of a 5′ flanking maize DNA sequence of SEQ ID NO: 1,or a sufficient length of a transgene portion of the DNA sequence of SEQID NO: 2 and a sufficient length of a 3′ flanking maize DNA sequence ofSEQ ID NO: 2. These primers are useful in PCR methods to provide a DNAamplicon product that is diagnostic for Event LY038 and its progeny. PCRprimers homologous or complementary to any suitable length of SEQ IDNOs: 1 and 2 that can produce an amplicon or probe that is diagnosticfor Event LY038 are another aspect of the present invention. Forexample, without limitation, preferred primers that are diagnostic forEvent LY038 include those having at least about 18 contiguousnucleotides of either of the sequences of SEQ ID NO: 3 or 4. Theamplicons produced using DNA primers that are diagnostic for Event LY038and its progeny are an aspect of this invention. A preferred amplicondiagnostic for Event LY038 has the sequence of SEQ ID NO: 6.

Another aspect of the present invention provides methods of detectingthe presence or absence of DNA corresponding to Event LY038 in a sample.Such methods comprise obtaining DNA from a maize plant, seed, or tissue,contacting the sample DNA with a PCR primer set, performing PCR anddetecting the presence or absence of an amplicon. Preferred PCR primersdiagnostic for Event LY038 include oligonucleotide primers having thesequence of SEQ ID NOs: 3 and 4, which produce an LY038 event-specificamplicon having the sequence of, for example, SEQ ID NO: 6, which isdetectable by an LY038 event-specific probe having the sequence of, forexample, SEQ ID NO: 5.

Hybridization of a probe indicating the presence of Event LY038 to anamplicon comprising DNA specific to Event LY038 may be detected by anysuitable means available to nucleic acid manipulation arts, includingTaqMan® assays, Southern blot methods among other methods known to thoseof ordinary skill in the art of molecular biology. One skilled in theart would know that the detecting of the amplicon may be carried out bymeans of detection that do not involve hybridization of a probe to anamplicon, such as acrylamide gel or agarose gel analyses. One skilled inthe art would also know that both the length and sequence of both theprimer and probe may be varied from the exemplified sequences presentedin SEQ ID NOs: 3, 4, and 5, and still produce a PCR amplicon, oramplicon and probe set, that is diagnostic for Event LY038.

In another aspect, the present invention provides a method for producingprogeny plants comprising Event LY038 DNA. The progeny plants may beinbred or hybrid plants. In a further application, the present inventionprovides a method for performing marker-assisted breeding for EventLY038 DNA. According to another aspect of the present invention, astably transformed maize plant comprising Event LY038 DNA and furthercomprising increased lysine in the kernel or parts thereof is provided.

The present invention further relates to a DNA detection kit comprisingat least one DNA molecule of sufficient length of contiguous nucleotideshomologous or complementary to SEQ ID NO: 1 or 2, that functions as aDNA primer or probe specific for Event LY038 or its progeny.

This present invention further relates to the plants and seeds andprocessed products thereof of high lysine maize (Zea mays) comprisingEvent LY038 and the progeny derived thereof having representative seeddeposited as ATCC Accession No. PTA-5623. Additionally provided by thepresent invention is a maize plant or a part thereof, including, forexample, pollen or seed, produced by growing a plant that comprisesEvent LY038 DNA. The maize plant and seed comprising Event LY038 DNA forwhich the DNA primer molecules of the present invention are usefullyemployed for detection of the event-specific sequences are furtheraspects of the invention.

A processed product of LY038 Event comprises a part of a maize kernel,for example, the endosperm. A maize meal of the present invention can bemade from the kernel comprising the transgenic DNA molecule of LY038,wherein the meal is high in lysine relative to other maize meals notcontaining the DNA molecule.

The foregoing and other aspects of the present invention will becomemore apparent from the following detailed description, examples, andaccompanying figures. The following examples are included to demonstrateexamples of certain preferred embodiments of the present invention. Itshould be appreciated by those of skill in the art that the techniquesdisclosed in the examples that follow represent approaches the inventorshave found function well in the practice of the present invention, andthus can be considered to constitute examples of preferred modes for itspractice. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plasmid map of pMON55221.

FIG. 2A is a schematic of exogenous DNA insertion Event LY038. ExogenousDNA and pertinent base pairs are indicated by italic font, maize genomicDNA and pertinent base pairs are indicated by regular font.

FIG. 2B is the sequence at the 5′ junction comprising SEQ ID NO. 5 forEvent LY038.

FIG. 2C is the sequence at the 3′ junction comprising SEQ ID NO. 11 forEvent LY038.

BRIEF DESCRIPTION OF SEQUENCES

Molecules having defined sequences used in the context of the presentinvention are set forth in the Sequence Listing filed concomitantly withthis application. A summary of the Sequence Listing follows:

SEQ ID NO: 1 is a 1961 base pair (bp) polynucleotide sequence of the 5′DNA comprising the maize genomic portion (bp 1–1781) flanking the 5′side of the insertion site and transgene insert portion (bp 1782–1961)of the LY038 event DNA.

SEQ ID NO: 2 is an 867 bp polynucleotide sequence of the 3′ DNAcomprising the 3′ maize genomic portion (bp 201–867) flanking the 3′side of the insertion site and transgene insert sequence (bp 1–200) ofthe LY038 Event DNA.

SEQ ID NOs: 3 and 4 are polynucleotide sequences of PCR primers usefulfor producing an amplicon diagnostic for Event LY038 DNA.

SEQ ID NO: 5 is a polynucleotide sequence of an oligonucleotide probeuseful for hybridizing to an amplicon for detecting Event LY038 DNA.

SEQ ID NO: 6 is a polynucleotide sequence of an amplicon diagnostic forEvent LY038 DNA.

SEQ ID NO: 7 is a polynucleotide sequence of a maize globulin 1 promoter(bp 48 to 1440; Kriz, Biochem. Genet., 27:239–251, 1989; Belanger andKriz, Genetics, 129:863–872, 1991; U.S. Pat. No. 6,329,574, incorporatedherein by reference in its entirety), a rice actin 1 intron (bp 1448 to1928; McElroy et al., Plant Cell, 2:163–171, 1990), a maize DHDPSchloroplast transit peptide (bp 1930 to 2100; Frisch et al., Mol. Gen.Genet., 228:287–293, 1991), a Corynebacterium DHDPS gene (bp 2101 to3003; Bonnassie et al., Nucleic Acids Research, 18:6421, 1990); Richaudet al., J. Bacteriol., 166:297–300, 1986), a maize globulin 1 3′untranslated region (bp 3080 to 4079; Belanger and Kriz, supra), and alox P site (bp 4091 to 4124; Russell et al., Mol. Gen. Genet.,234:45–59, 1992).

SEQ ID NO: 8 is a polynucleotide sequence of a Corynebacterium DHDPSgene (Bonnassie et al., Nucleic Acids Research, 18:6421, 1990; Richaudet al., J. Bacteriol., 166:297–300, 1986).

SEQ ID NO: 9 is a 1736 base pair polynucleotide sequence of additionalmaize genomic DNA flanking the 5′ side of the insertion site of theLY038 event (see FIG. 2A).

SEQ ID NO: 10 is a 359 base pair polynucleotide sequence of additionalmaize genomic DNA flanking the 3′ side of the insertion site of theLY038 event (see FIG. 2A).

SEQ ID NO: 11 is a 20 base pair polynucleotide sequence consisting of 10contiguous nucleotide of transgene insert DNA and 10 contiguousnucleotides of maize genomic DNA of the junction sequence illustrated inFIG. 2C.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “exogenous DNA” refers to DNA that does not naturallyoriginate from the particular construct, cell, or organism in which thatDNA is found. Exogenous DNA may include a DNA or RNA sequence native toa genome but in a new location in the genome or linked to other sequenceelements not naturally associated with the exogenous DNA in its nativestate. Recombinant DNA constructs used for transforming plant cellscomprise exogenous DNA and usually other elements as discussed below. Asused herein “transgene” means an exogenous DNA that has beenincorporated into a host genome or is capable of autonomous replicationin a host cell and is capable of causing the expression of one or morecellular products. Exemplary transgenes provide the host cell or plantsregenerated therefrom, with a novel phenotype relative to thecorresponding non-transformed progenitor cell or plant, or acorresponding transformed progenitor cell or plant comprising othertransgenes but not the particular transgene in question. Transgenes maybe directly introduced into a plant by genetic transformation, or may beinherited from a plant of any previous generation that was transformedwith the exogenous DNA.

As used herein, “gene” or “coding sequence” means a DNA sequence fromwhich an RNA molecule is transcribed. The RNA may be an mRNA thatencodes a protein product, an RNA that functions as an anti-sensemolecule, or a structural RNA molecule such as a tRNA, rRNA, snRNA, orother RNA. As used herein “expression” refers to the combination ofintracellular processes, including transcription and translation, bywhich a DNA molecule, such as a gene, is employed to produce apolypeptide or an RNA molecule. An exemplary coding sequence is aCorynebacterium dihydrodipicolinate synthase gene (DHDPS; Bonnassie etal., Nucleic Acids Research, 18:6421, 1990; Richaud et al., J.Bacteriol., 166:297–300, 1986; bp 2101 to 3003 of SEQ ID NOs: 7 and 8),useful for the production of maize kernels with increased lysine. Amaize plant, transformed to contain and express a Corynebacterium DHDPSgene resulting in increased lysine in kernel tissue, is also referred toas a high lysine maize plant.

As used herein, “promoter” means a region of DNA sequence that isessential for the initiation of transcription of DNA, resulting ingeneration of an RNA that is complementary to the transcribed DNA; thisregion may also be referred to as a “5′ regulatory region.” Promotersare located upstream of the coding sequence to be transcribed and haveregions that act as binding sites for RNA polymerase and have regionsthat work with other factors to promote RNA transcription. Useful plantpromoters include those that are constitutive, inducible,tissue-specific, temporally regulated, circadian in regulation, droughtinducible, stress inducible, developmentally regulated, cold inducible,light inducible, and the like. Of particular importance to the presentinvention is an embryo-specific promoter, such as, without limitation,the maize globulin 1 promoter (Kriz, Biochem. Genet., 27:239–251, 1989;Belanger and Kriz, Genetics, 129:863–872, 1991; bp 48 to 1440 of SEQ IDNO: 7).

As is well known in the art, recombinant DNA constructs typically alsocomprise other regulatory elements in addition to a promoter, such asbut not limited to 3′ untranslated regions (such as polyadenylationsites or transcriptional termination signals), transit or signalpeptides, introns, and marker genes elements. A 3′ untranslated region(3′ UTR) useful in the practice of the present invention is the globulin1 3′ UTR (Kriz, Biochem. Genet., 27:239–251, 1989; Belanger and Kriz,Genetics, 129:863–872, 1991; bp 3080 to 4079 of SEQ ID NO: 7). Aparticularly useful transit peptide is the maize DHDPS transit peptide(Frisch et al., Mol. Gen. Genet., 228:287–293, 1991; bp 1930 to 2100 ofSEQ ID NO: 7). An intron useful in the context of the present inventionis the rice actin 1 intron 1 (McElroy et al., Plant Cell, 2:163–171,1990; bp 1448 to 1928 of SEQ ID NO: 7).

As used herein, the term “maize” means Zea mays, also known as corn, andincludes all plant varieties that can be bred with maize, including wildmaize species. Methods and compositions for transforming plants byintroducing an exogenous DNA into a plant genome in the practice of thisinvention can include any of the well-known and demonstrated methods. Todate, microparticle- and Agrobacterium-mediated gene delivery are thetwo most commonly used plant transformation methods.Microparticle-mediated transformation refers to the delivery of DNAcoated onto microparticles that are propelled into target tissues byseveral methods. Agrobacterium-mediated transformation is achievedthrough the use of a genetically engineered soil bacterium belonging tothe genus Agrobacterium. Several Agrobacterium species mediate thetransfer of a specific DNA known as “T-DNA,” which can be geneticallyengineered to carry any desired piece of DNA into many plant species.Preferred methods of plant transformation are microprojectilebombardment as illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318;5,538,880; 6,160,208; 6,399,861; and 6,403,865; andAgrobacterium-mediated transformation as illustrated in U.S. Pat. Nos.5,635,055; 5,824,877; 5,591,616; 5,981,840; and 6,384,301, all of whichare incorporated herein by reference.

As used herein a “transgenic” organism is one whose genome has beenaltered by the incorporation of foreign genetic material or additionalcopies of native genetic material, e.g. by transformation orrecombination. The transgenic organism may be a plant, mammal, fungus,bacterium, or virus. As used herein “transgenic plant” means a stablytransformed plant or progeny plant of any subsequent generation derivedtherefrom, wherein the DNA of the plant or progeny thereof contains anintroduced exogenous DNA not originally present in a non-transgenicplant of the same strain. The transgenic plant may additionally containsequences that are native to the plant being transformed, but whereinthe exogenous DNA has been altered in order to alter the level orpattern of expression of the gene.

As used herein, a “stably” transformed plant is a plant in which theexogenous DNA is heritable. The exogenous DNA may be heritable as afragment of DNA maintained in the plant cell and not inserted into thehost genome. Preferably, the stably transformed plant comprises theexogenous DNA inserted into the chromosomal DNA in the nucleus,mitochondria, or chloroplast, most preferably in the nuclear chromosomalDNA.

As used herein a “R_(o) transgenic plant” is a plant that has beendirectly transformed with an exogenous DNA or has been regenerated froma cell or cell cluster that has been transformed with an exogenous DNA.As used herein “progeny” means any subsequent generation, including theseeds and plants therefrom, which is derived from a particular parentalplant or set of parental plants; the resultant progeny line may beinbred or hybrid. Progeny of a transgenic plant of this presentinvention can be, for example, self-crossed, crossed to a transgenicplant, crossed to a non-transgenic plant, and/or back crossed.

The seeds of the plants of this present invention can be harvested fromfertile transgenic plants and be used to grow progeny generations ofplants of this present invention, including a hybrid plant linecomprising the exogenous DNA of Event LY038, which provides the benefitof increased lysine in the maize kernel. The maize kernel may beprocessed into meal and oil products or the kernel can fed to animalswithout processing. The meal product in particular contains an enhancedagronomic trait, increased lysine. The present invention contemplates amaize meal with increased lysine relative to other maize meals, whereinthe maize meal comprises the exogenous DNA of Event LY038.

The term “Event LY038 DNA” refers to a DNA segment comprising anexogenous DNA of SEQ ID NO:7 inserted into a particular location in thegenome, as shown in FIG. 2A, and adjacent flanking genomic DNA thatwould be expected to be transferred to a progeny plant from a parentplant containing the exogenous DNA. More specifically, Event LY038 DNAalso refers to each of the DNA regions that include an interface of thegenomic DNA and the inserted exogenous DNA in the genome of the R_(o)transformant, e.g., a region around one interface where the 5′ end is ingenomic DNA and the 3′ end is in exogenous DNA, as depicted by SEQ IDNOs: 1, 2, 5, 6 and 11. In addition, the sequence of the exogenous DNAcomprising an event DNA may be altered while resident in its particularlocation in a host genome, e.g., a portion of the sequence may bechanged, deleted or amplified and still constitute said event DNAproviding said exogenous DNA continues to reside in the same location inthe genome and when expressed in a plant provides increased lysinelevels.

A transgenic “event” is produced by transformation of a plant cell withan exogenous DNA construct, the regeneration of a plant resulting fromthe insertion of the exogenous DNA into the genome of the plant, andselection of a particular plant characterized by event DNA. Typically, anumber of plant cells are transformed, producing a population of plantsfrom which a particular plant is selected. The term “event” refers tothe original R_(o) transformant and progeny of the transformant thatinclude the exogenous DNA inserted into a particular and unique locationin the genome, i.e., event DNA. The term “event” also refers to progenyproduced by a sexual outcross, a self-cross, or repeated backcrossing,wherein at least one of the plants used in the breeding are of anygeneration of the original R_(o) transformant containing event DNA.

Thus, a transgenic “event” is a plant comprising and defined by an“event DNA.” In this way, “Event LY038” comprises “LY038 Event DNA.” Aplant may comprise two or more different event DNAs and thus comprisetwo or more different events. In addition, a plant lacking a giventransgene event X does not comprise that event DNA X in question. EventDNA may be transferred from plant to plant, generation to generation, byany breeding scheme, method, or tool known to those of skill in the artof maize breeding.

Transformation of plants typically utilizes a selectable marker andselection method to distinguish the transformed cells of the culturefrom the non-transformed cells. In some instances the selectable markergene remains in the transgenic plant; in other instances it is desirableto remove the selectable marker gene or other sequences introduced inthe exogenous DNA. Homologous recombination is one method useful for thedeletion of marker genes residing within a transgenic plant (U.S. Pat.No. 6,580,019, incorporated herein by reference in its entirety).Another useful tool for removing sequences from a plant involves the useof site-specific recombinase enzymes and their respective site-specifictarget sites.

A number of different site-specific recombinase systems could beemployed in accordance with the instant invention, including, but notlimited to, the Cre/lox system of bacteriophage P1, and the FLP/FRTsystem of yeast. The bacteriophage P1 Cre/lox and the yeast FLP/FRTsystems constitute two particularly useful systems for site-specificintegration or excision of transgenes. In these systems, a recombinase(Cre or FLP) will interact specifically with its respectivesite-specific recombination sequence (lox or FRT, respectively) toinvert or excise the intervening sequences. The sequence for each ofthese two systems is relatively short (34 bp for lox and 47 bp for FRT)and therefore, convenient for use with transformation vectors. TheFLP/FRT and Cre/lox recombinase systems have been demonstrated tofunction efficiently in plant cells. In a preferred embodiment, aCre/lox recombinase system is employed to removed selectable markersequences, particularly an NPT II marker gene (see FIG. 1) flanked bylox P recombination sites (bp 4091 to 4124 SEQ ID NO: 7; Russell et al.,Mol. Gen. Genet., 234:45–59, 1992).

A transgenic plant, seed or parts thereof, that shows an enhanceddesired trait, e.g., “increased lysine,” is a plant comprising anexogenous DNA that imparts a desired, measurable change in a trait incomparison to a plant of substantially the same genotype that lacks thedesired exogenous DNA. Preferably, the enhanced desired trait ismeasured by comparing the trait in a transgenic plant with the exogenousDNA associated with the enhanced desired trait to the trait in a plantof substantially the same genotype but lacking that exogenous DNA. Sucha plant that lacks that exogenous DNA can be a natural wild-type plantor a transgenic plant, preferably of the same species as the transgenicplant. Preferably, the plant lacking the exogenous DNA is a siblinglacking the desired exogenous DNA of the plant comprising the desiredexogenous DNA. Such a sibling plant may comprise other exogenous DNAs.Increased lysine may be exhibited by the plant by accumulation ofincreased amounts of the amino acid in the kernel and may be measured byany suitable method, such as that of mass spectrophotometry or highperformance liquid chromatography of appropriately extracted tissue.

As used herein, a “probe” is an isolated oligonucleotide to which may beattached a detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, dye, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid. In the case of thepresent invention, such a probe is complementary to a strand of genomicDNA from Event LY038, e.g., genomic DNA from a maize plant or seed orother plant part of Event LY038. Probes according to the presentinvention are materials, including DNA, RNA, and polyamides, that bindspecifically to a target DNA sequence and can be used to detect thepresence of that target DNA sequence.

“Primers” are isolated oligonucleotides that can anneal to acomplementary target DNA strand by nucleic acid hybridization and thenbe extended along the target DNA strand by a polymerase, e.g., a DNApolymerase. As used herein, primers of the present invention are usedfor DNA amplification of a target nucleic acid sequence, for example, bythe polymerase chain reaction (PCR) and may also be referred to as “PCRprimers.”

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence specifically under the hybridization conditions orreaction conditions determined by a skilled artisan. This length may beany length that is of sufficient length to be useful in the detectionmethod of choice. Generally, about 11 nucleotides or more in length,preferably about 18 nucleotides or more, more preferably about 24nucleotides or more, and, most preferably, about 30 nucleotides or moreare used. Such probes and primers hybridize specifically to a target.Preferably, probes and primers according to the present invention havecomplete DNA sequence similarity of contiguous nucleotides with thetarget sequence, although probes differing from the target DNA sequenceand that retains the ability to hybridize to target DNA sequences may bedesigned by conventional methods. Methods for preparing and using probesand primers are known to those of skill in the art, using protocolspublished in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,1989, and the like.

Identification of the flanking genomic DNA sequences surrounding theinsertion site of transgenic events allow for the design of detectionmethods that are specific for a given transgenic event inserted into aparticular location in a genome. Such a detection method, which candifferentiate between the same or similar transgenes located indifferent insertion sites in a genome, is termed an event-specific DNAdetection method, e.g., an event-specific assay. Event-specific assays,for example, for glyphosate tolerant maize event nk603 have beendescribed (U.S. Patent Application Publication 2002/0013960,incorporated herein by reference in its entirety).

In a preferred embodiment, a nucleic acid probe of the present inventionspecifically hybridizes to the LY038 Event-specific amplicon having thenucleic acid sequence of SEQ ID NOs: 3–6, or complements thereof, mostpreferably SEQ ID NO: 5 or complements thereof. In another aspect of thepresent invention, a preferred nucleic acid probe molecule of thepresent invention shares between about 80%, preferably about 90%, morepreferably about 95%, even more preferably about 98%, and mostpreferably about 99% sequence identity with the nucleic acid sequenceset forth in one or more of SEQ ID NOs: 3–6, or complements or fragmentsthereof. Exemplary probes diagnostic for Event LY038 have the sequenceof SEQ ID NO: 6. Such probe molecules may be used by those of skill inthe art as markers in plant breeding methods to identify the progeny ofgenetic crosses. The hybridization of the probe to the target DNAmolecule can be detected by any number of methods known to those skilledin the art. These detection methods can include, but are not limited to,fluorescent tags, radioactive tags, antibody based tags, andchemiluminescent tags.

As used herein, “homologous” refers to a nucleic acid sequence that willspecifically hybridize to the complement of the nucleic acid sequence towhich it is being compared under high stringency conditions. Appropriatestringency conditions, including those conditions of time, temperature,and salt condition, that promote DNA hybridization are known to thoseskilled in the art. Both temperature and salt may be varied, or eitherthe temperature or the salt concentration may be held constant while theother variable is changed. In a preferred embodiment, a polynucleic acidof the present invention will specifically hybridize to one or more ofthe nucleic acid molecules set forth in SEQ ID NO: 1 or 2, orcomplements thereof or fragments of either under strong to moderatelystringent conditions. In a particularly preferred embodiment, a nucleicacid of the present invention will specifically hybridize to one or moreof the nucleic acid molecules set forth in SEQ ID NO: 1 or 2 orcomplements or fragments of either under high stringency conditions. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

As used herein, “amplicon” refers to the product of nucleic-acidamplification of a target nucleic acid sequence that is part of anucleic acid template. For example, to determine whether the maize plantresulting from a sexual cross contains transgenic event genomic DNA fromthe maize plant comprising exogenous LY038 DNA, DNA extracted from amaize plant tissue sample may be subjected to a nucleic acidamplification method using a DNA primer pair that includes a firstprimer derived from flanking sequence in the genome of the plantadjacent to the insertion site of inserted heterologous DNA, and asecond primer derived from the inserted heterologous DNA to produce anamplicon that is diagnostic for the presence of the event DNA. Theamplicon is of a length and has a sequence that is also diagnostic forthe event. The amplicon may range in length from the combined length ofthe primer pairs plus one nucleotide base pair, preferably plus about 20nucleotide base pairs, more preferably plus about 50 nucleotide basepairs, and even more preferably plus about 150 nucleotide base pairs andmore depending on the method used to detect the amplicon. Alternatively,a primer pair can be derived from flanking sequence on both sides of theinserted DNA so as to produce an amplicon that includes the entireinsert nucleotide sequence. A member of a primer pair derived from theplant genomic sequence may be located a distance from the inserted DNAsequence. This distance can range from one nucleotide base pair up tothe limits of the amplification reaction, or about 20,000 nucleotidebase pairs. The use of the term “amplicon” specifically excludes primerdimers that may be formed in the DNA thermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). The sequence of the heterologous DNAinsert or flanking DNA sequence from Event LY038 can be verified (andcorrected if necessary) by amplifying such sequences from DNA extractedfrom the ATCC deposit Accession No. PTA-5623 seed or plants using DNAprimers derived from the sequences provided herein followed by standardDNA sequencing of the PCR amplicon or of the cloned DNA.

Primers and probes based on the flanking genomic DNA and insertsequences disclosed herein can be used to confirm (and, if necessary, tocorrect) the disclosed DNA sequences by conventional methods, e.g., byre-cloning and sequencing such DNA molecules.

Amplicons produced by amplification methods may be detected by aplurality of techniques, including but not limited to gel basedanalyses, genetic bit analysis (Nikiforov et al., Nucleic Acid Res.,22:4167–4175, 1994), pyrosequencing (Winge, M., Pyrosequencing—a newapproach to DNA analysis, (2000), Innovations in PharmaceuticalTechnology, vol 00, 4, p 18–24), fluorescence polarization (Chen et al.,Genome Res., 9:492–498, 1999), and molecular beacons (Tyangi et al.,Nature Biotech., 14:303–308, 1996).

Of particular interest to the present invention is detection by Taqman®assay (available from Applied Biosystems, Foster City, Calif.). Taqman®assay is a method of detecting and quantifying the presence of a DNAsequence that is well known in the art, and is fully described in theinstructions provided by the manufacturer. This method involves the useof PCR amplification and detection of the amplification product byhybridization using a special FRET oligonucleotide probe. The FREToligonucleotide probe is designed to have a 5′ fluorescent reporter dyeand a 3′ quencher dye covalently linked to the 5′ and 3′ ends of theprobe. The probe is designed to overlap the junction of the genomic DNAand inserted DNA. The FRET probe and PCR primers (one primer in theexogenous transgene DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

PCR primers preferable for use with Taqman® assay are designed (a) tohave a length in the size range of 18 to 25 bases and matching sequencesin the flanking genomic DNA and the transgene insertion, (b) to have acalculated melting temperature in the range of about 57 to about 60° C.,e.g., corresponding to an optimal PCR annealing temperature of about 52to about 55° C., and (c) to produce a product that includes the junctionbetween the flanking genomic DNA and the transgene insertion and has alength in the size range of about 75 to about 250 base pairs. The PCRprimers are preferably located on the locus so that the junctionsequence is at least one base away from the 3′ end of each PCR primer.The PCR primers must not contain regions that are extensively self- orinter-complementary.

FRET probes are designed to span the sequence of the junction sequence.In a preferred embodiment, the FRET probes will have incorporated attheir 3′ end a chemical moiety that, when the probe is annealed to thetemplate DNA, binds to the minor groove of the DNA, thus enhancing thestability of the probe-template complex. The probes preferably have alength in the range of about 12 to about 17 bases, and with the 3′ minorgroove binding moiety, have a calculated melting temperature of about 5to about 7° C. above that of the PCR primers. Probe design is disclosedin U.S. Pat. Nos. 5,538,848; 6,084,102; and 6,127,121.

Another assay that employs the sequences of the present invention isthat of a zygosity assay. A zygosity assay is useful for determining ifa plant comprising an event is homozygous for the event DNA, that iscomprising the exogenous DNA in the same location on each chromosome ofa chromosomal pair, or heterozygous for an event DNA, that is comprisingthe exogenous DNA on only one chromosome of a chromosomal pair. In oneembodiment, a three primer assay is employed wherein primer 1 hybridizesand extends specifically from the inserted exogenous DNA, primer 2hybridizes and extends specifically from the DNA flanking the 5′ side ofthe inserted exogenous DNA, and primer 3 hybridizes and extendsspecifically from the DNA flanking the 3′ side of the inserted exogenousDNA. The three primers are diagnostic for the event. Typically, theexogenous DNA is of such a size, e.g., about 3 to about 7 kilobases ormore, such that primer 1 and primer 3 no longer produce an amplicon inthe PCR reaction. When the three primers are mixed together in a PCRreaction with DNA extracted from a plant homozygous for a given event, asingle amplicon is produced by primer 1 and primer 2, the size andsequence of which will be indicative of and diagnostic for the eventDNA. When the three primers are mixed together in a PCR reaction withDNA extracted from a plant that does not comprise the given event, asingle amplicon is produced by primer 1 and primer 3, the size andsequence of which will be indicative of and diagnostic for maize genomicDNA lacking an exogenous DNA. When the three primers are mixed togetherin a PCR reaction with DNA extracted from a plant that is heterozygousfor a given event, 2 amplicons are produced: 1) an amplicon is producedby primer 1 and primer 3, the size and sequence of which will beindicative of and diagnostic for maize genomic DNA lacking an exogenousDNA, and 2) an amplicon is produced by primer 1 and primer 2, the sizeand sequence of which will be indicative of and diagnostic for the eventDNA. Methods of detecting the various amplicons produced by the zygosityassay are known to those of skill in the art and include, but are notlimited to, gel electrophoresis, TaqMan® assays, Southern blot, InvaderTechnology, sequencing, molecular beacons, pyrosequencing, and the like.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for identification of maize Event LY038 DNA in a sample andcan be applied to methods for breeding maize plants containing EventLY038 DNA. The kits contain DNA sequences that are useful as primers orprobes and that are homologous or complementary to any portion of SEQ IDNO: 1 or 2, or to DNA sequences homologous or complementary to DNAcontained in any of the transgene genetic elements of pMON55221 (FIG. 1)that have been inserted into a maize plant genome to form Event LY038(FIG. 2). These DNA sequences can be used in DNA amplification methods(PCR) or as probes in polynucleic acid hybridization methods, i.e.,Southern analysis, or Northern analysis. The DNA molecule (SEQ ID NO:7)contained in the LY038 Event genome comprises the heterologous transgenegenetic elements that includes a maize globulin 1 promoter (P-ZM glob1),a rice actin 1 intron (I-Os actin), a maize dihydrodipicolinate synthasechloroplast transit peptide encoding-DNA molecule (Zm DHDPS CTP), aCorynebacterium dihydrodipicolinate synthase-encoding DNA molecule(DHDPS), a maize globulin 1 3′ untranslated region (T-Zm glob1), and alox P site and can be used as a template for DNA amplification, or toselect homologous or complementary DNA molecules that can be used as aDNA primer or probe in a DNA detection method. The present inventioncontemplates that one skilled in the art of DNA detection can select oneor more DNA molecules homologous or complementary to the transgenic DNAof SEQ ID NO: 7 that is useful in a method to detect the transgenic DNAin the genome of LY038 and progeny thereof.

The following examples are included to demonstrate examples of certainpreferred embodiments of the present invention. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches the inventors have foundfunction well in the practice of the present invention, and thus can beconsidered to constitute examples of preferred modes for its practice.However, those of skill in the art should, in light of the presentdisclosure, appreciate that many changes can be made in the specificembodiments that are disclosed and still obtain a like or similar resultwithout departing from the spirit and scope of the invention.

EXAMPLE 1

Preparation Of Transgenic Plants

Immature embryos of maize line H99 were isolated for transformation.Cassette DNA isolated from vector pMON55221 (see FIG. 1) comprising amaize globulin 1 promoter (Kriz (1989), supra; Belanger and Kriz (1991),supra; U.S. Pat. No. 6,329,574, incorporated herein by reference in itsentirety; bp 48 to 1440 of SEQ ID NO: 7), a rice actin 1 intron (McElroyet al. (1990), supra; bp 1448 to 1928 of SEQ ID NO: 7), a maize DHDPSchloroplast transit peptide encoding DNA molecule (Frisch et al. (1991),supra; bp 1930 to 2100 of SEQ ID NO: 7), a Corynebacteriumdihydrodipicolinate synthase encoding DNA molecule (Bonnassie et al.(1990), supra; Richaud et al. (1986), supra; bp 2101 to 3003 of SEQ IDNO: 7), a maize globulin 1 3′ untranslated region (Belanger and Kriz(1991), supra; bp 3080 to 4079 of SEQ ID NO: 7), a lox P site (U.S. Pat.No. 5,658,772, specifically incorporated herein by reference in itsentirety; bp 4091 to 4124 of SEQ ID NO: 7), as well as a 35S promoter(Kay et al., Science, 236:1299–1302, 1987; U.S. Pat. No. 5,164,316), anNPTII selectable marker encoding DNA molecule (Potrykus et al. (1985),supra), a nos 3′ UTR (Fraley et al., Proc. Natl. Acad. Sci. (U.S.A.),80:4803–4807 (1983), supra), and a lox P site (U.S. Pat. No. 5,658,772,specifically incorporated herein by reference in its entirety) wasadhered to gold particles. Microprojectile bombardment was used tointroduce the exogenous DNA to the immature maize embryos using methodsknown to those of skill in the art. Transformed cells were selectedusing a kanamycin selection scheme. Kanamycin resistant calli wereobtained and regenerated into several fertile, R_(o) transgenic plantsusing standard methods.

EXAMPLE 2

Breeding Scheme and Lysine Analysis

Table 1 summarizes the breeding scheme and free lysine data used for thedevelopment of maize Event LY038, which exhibits high lysine in maizekernel tissue. Plants produced by the transformation method described inExample 1 were initially screened for the presence or absence of theCorynebacterium DHDPS sequence using PCR. Taqman® assay technology wasused to determine the number of copies of transgenic insertions. Plantsthat comprised a DNA molecule having the Corynebacterium DHDPS sequence,operably linked as described in Example 1 and FIG. 1, were allowed toreach maturity and to produce F_(1A) seed. For F_(1A) seed production,the primary transgenic R_(o) plants were crossed with a non-transgenicelite maize inbred line.

The F_(1A) plants were screened for the presence or absence of the NPTIIsequence as evidenced by using a field scorable kanamycin resistancetest. Insensitivity to kanamycin indicated that a plant comprised andwas expressing the NPTII marker gene as shown in FIG. 1 and described inExample 1; these plants are referred to hereafter as NPTII+.

NPTII+ plants were crossed with a transgenic maize line expressing abacterial Cre recombinase to produce F_(1B) seed. The levels of freelysine in a sample of the resulting F_(1B) seed collected from each earwas determined. Sibling F_(1B) kernels exhibiting greater than about1000 ppm free lysine were advanced to a field nursery. F_(1B) progenyplants were assayed by PCR and/or Southern blot to determine thepresence or absence of the DHDPS gene sequence, the Cre recombinasecoding sequence and the NPTII selectable marker gene sequence. The NPTIIselectable marker gene was flanked by lox P sites (recombination sites)and as such, the activity of the Cre recombinase resulted in theexcision of the NPTII coding sequence. Plants comprising the DHDPS andCre recombinase sequences and lacking the NPTII sequences, referred tohereafter as marker-excised plants, were allowed to self-pollinate toproduce F_(2A) seed. Free lysine in the positive and negative F_(2A)seed was determined.

Having obtained F_(2A) seed comprising the exogenous DHDPS gene ofinterest and lacking the NPTII selectable marker gene, it was nownecessary to breed away the Cre recombinase sequences. F_(2A) seed frommarker-excised plants were planted in the field and once again assayedby PCR and/or Southern blot to determine the presence or absence of theDHDPS gene sequence, the Cre recombinase coding sequence and the NPTIIselectable marker gene sequence. Plants comprising the DHDPS sequenceand lacking sequences for both Cre recombinase and NPTII were selectedas “positive” plants. Sibling plants lacking the DHDPS, Cre recombinase,and NPTII sequences were selected as “negative” plants to serve asnegative controls. Plants comprising the DHDPS sequence wereself-pollinated to create F₃ seed and advanced to the next generation inthe field. Likewise, negative plants lacking the DHDPS, Cre recombinase,and NPTII sequences were self-pollinated to create F₃ seed. Free lysinein the positive and negative F₃ seed was determined.

Positive plants were grown in the field from F₃ seed. A single plant,determined by Taqman® assay to be homozygous, was selected anddesignated as LY038. F₃ plants were either A) self-pollinated to produceF₄₋₃₈ ears, or were B) crossed with an inbred line to produce F_(1-38A)ears of both positive and negative selections. Free lysine in thepositive and negative F₄₋₃₈ seed was determined.

F_(1-38A) seed of Event LY038 was field grown and allowed to selfpollinate to produce F_(2B) seed for which free lysine was determined.

F₄₋₃₈ seed of Event LY038 was field grown to produce F₄₋₃₈ plants andeither A) self pollinated to produce F₅₋₃₈ seed, or B) crossed to aninbred line to produce hybrid F_(1-38B) seed used for agronomicevaluation. F₅₋₃₈ seed of Event LY038 was field grown and either A) selfpollinated to produce F₆₋₃₈ seed, or B) crossed to an inbred line maizevariety to produce additional hybrid F_(2C) seed used for additionalagronomic evaluation.

Deposits of the Monsanto Company maize seed of Event LY038 disclosedabove was generated through the self-pollinating of F₅₋₃₈ plants toproduce F₆₋₃₈ seed and the self-pollinating of F₆₋₃₈ plants to produceF₇₋₃₈ seed. The American Type Culture Collection (Manassas, Va.)accession number for Event LY038 is PTA-5623.

Free lysine accumulation was monitored during the development of EventLY038 maize lines. The lysine accumulation values are summarized inTable 1 and represent the quantity of free lysine present in the maturegrain on a dry weight basis, in parts-per-million.

Different methods are useful to evaluate the lysine content of maturekernels comprising Event LY038. Other methods known the art that areuseful to detect and quantitate lysine are contemplated by the inventorsof the present invention to provide similar findings of an increase inlysine content of seed of LY038.

Liquid chromatography-mass spectrophotometry/mass spectrophotometry(LC-MS/MS) was used to analyze free lysine in the maize kernels of EventLY038. Individual mature maize kernel samples of Event LY038 were firstweighed, ground to a fine, homogeneous powder and extracted with anextraction solvent comprising methanol, water, and formic acid. Insituations where kernels were bulked, approximately 30 mg of groundpowder was used. Both liquid chromatography andmultiple-reaction-monitoring (MRM) mass spectrometric techniques wereused to separate lysine in the sample extract. After the separation,lysine was quantified using its mass spectrometry peak area against itscorresponding standard calibration curve which was prepared using adeuterated d₄-lysine internal standard (IS).

In another method, lysine content of maize kernels was based uponevaluation of the free amino acids by high performance liquidchromatography (HPLC). Individual maize kernels or pools of kernels ofEvent LY038 were ground to a fine, homogenous powder as described, andin this instance, approximately 30 mg of powder was used for analysis.Amino acids were extracted with 5% trichloroacetic acid and amino aciddetection was achieved through a pre-column primary amine derivatizationwith o-phthalaldehyde (OPA). The resulting amino acid adduct, anisoindole, is hydrophobic and possesses excellent fluorescencecharacteristics, which can then be detected on a fluorescence detector.Using reverse-phase chromatography, separation is achieved through thehydrophobicity of the R-groups located on each amino acid. To helpstabilize the fluorophor, a thiol is added such as 2-mercaptoethanol(SHCH₂CH₂OH) or 3-mercaptopropionic acid (SHCH₂CH₂COOH).

TABLE 1 Breeding scheme and lysine analysis used to identify high lysinemaize Event LY038. Negative LY038 Control Plant Molecular & Seed LysineLysine Generation Field analyses Pollination Produced efficacy*efficacy* R₀ plant PCR; TaqMan Crossed with F_(1A) seed ND ND (Inbred A)copy number inbred line determination F_(1A) plant NPTII field Crossedwith F_(1B) seed +++ − assay Cre recombinase line F_(1B) plant PCR forSelf pollinate; F_(2A) seed + − DHDPS, Cre positives and recombinase &negatives NPTII; Southern selected and Blot analysis maintained F_(2A)plants PCR; Southern Self pollinate F₃ seed + − (pos & neg) Blotanalysis F₃ plants PCR Self pollinate; F_(4–38) and +^(i) − (pos & neg)cross to F_(1–38A) inbred lines; hybrid seed select line LY038 F_(1–38A)plants Self pollinate F_(2B) seed ++ − (F3 plants × inbred) F_(4–38)plants Event-specific Self pollinate F_(5–38) and +^(i) − (pos & neg)PCR and cross to F_(1–38B) inbred lines hybrid seed F_(1–38B) plantsSelf pollinate F_(2C) seed ++ − (F_(4–38) plants × inbred) *representsppm free lysine in mature kernels comprising Event LY038 ND = notdetermined − indicates less than about 400 ppm free lysine + indicatesabout 1000 to about 1200 ppm free lysine ++ indicates about 1200 toabout 1400 ppm free lysine +++ indicates greater than about 1400 ppmfree lysine i = inbred kernel data

Based on the experiments described here, free lysine in maize kernelsthat contain the LY038 construct increased between about 200% (e.g.,F_(1B), among others) and nearly 300% (e.g., F_(1A)). Intermediateincreases in free lysine were also observed (e.g., F_(1-38A)).

EXAMPLE 3

Determination of Flanking Sequence

Genomic DNA was isolated from maize plants designated as LY038 and usedin experiments to determine the maize genomic sequence flanking thetransgenic DNA insert. Three different methods for determining flankingsequences and the sequence of the junction between the genomic flankingsequence and transgenic insert were used: tail PCR and the GenomeWalker™ kit from ClonTech (catalog number K1807-1, ClonTechLaboratories, Palo Alto, Calif.), and inverse PCR.

Tail PCR is a method for isolating genomic DNA sequence flanking a knowninserted sequence which employs degenerative primers and a biotincapture step. A primer complementary to the exogenous DNA is used inprimary PCR reactions together with a variety of degenerative primers.Typically, primers specific for the exogenous DNA and the degenerateprimers were used in pairs and not in pools. The degenerate primershybridize to some degree to the maize genomic sequence flanking theinserted DNA to allow for the generation of PCR amplicons. The primaryPCR amplicons are mixed with a biotin labeled primer complementary tothe transgene portion of the amplicon and allowed to anneal. Theamplicons annealed to the biotin primers were captured usingstreptavidin and unbound amplicons were washed away. The annealedamplicons were subjected to secondary PCR reactions utilizing a nestedprimer which was complementary to the exogenous DNA portion of theamplicon and a variety of degenerate primers. The PCR amplicons of thesecondary PCR reaction were subjected to agarose gel electrophoresis andbands were cut from the gel and isolated. The isolated PCR ampliconswere sequenced. The sequence of the 3′ flanking genomic DNA of EventLY038 was identified using tail PCR and sequencing.

The Genome Walker method for isolation of flanking DNA was carried outaccording to manufacturer's suggested conditions. Both tail PCR and theGenome Walker Kit were used to identify the sequence of the 5′ flankingDNA of Event LY038. For Genome Walker, products of the restrictionenzyme ScaI were amplified to produce amplicons useful for identifyingthe 5′ flanking genomic DNA sequence of Event LY038.

Use of the tail PCR and Genome Walker methods generated several hundredbase pairs or more of DNA sequence flanking the insertion site of theDNA construct in Event LY038. Inverse PCR and bioinformatic analysis andcomparison to maize genomic DNA sequence databases were used to obtainadditional genomic DNA flanking these events. Using the combinedmethods, the flanking sequences are identified in the sequence of SEQ IDNOs: 1, 2, 9, and 10. A maize plant is an aspect of the presentinvention when that maize plant contains within its genome a DNAmolecule that can be used as a template in a DNA amplification reactionto provide an amplicon comprising a junction DNA molecule described inthe present invention, wherein the junction DNA molecule is diagnosticfor corn Event LY038 DNA in a DNA sample extracted from a maize tissuesample.

EXAMPLE 4

Event Specific Primer and Probe Assay Information

For each event, PCR primers and probes useful in a Taqman® assay weredesigned, namely SEQ ID NOs: 3 and 4. Using the PCR primers having thesequence of SEQ ID NOs: 3 and 4, in a Taqman® assay resulted in anamplicon diagnostic for Event LY038; the amplicon has the sequence ofSEQ ID NO: 6, and the probe useful for the detection of this ampliconhas the sequence of SEQ ID NO: 5. When the primers and probes weresubjected to the PCR conditions outlined in Table 2, a fluorescentsignal indicated that amplicons were produced that were detected by theprobe. By inclusion of the appropriate control samples, for example,various negative and positive DNA controls, it was shown that the PCRprimers and probes were specific for the intended event.

In addition to the primer and probe set, any primer and probe setsderived from SEQ ID NO: 1 or 2, specific for Event LY038 DNA that whenused in a PCR amplification reaction produce a DNA amplicon diagnosticfor Event LY038 DNA are an aspect of the present invention and arereadily prepared by those of skill in the art. PCR conditions to producea Taqman® assay diagnostic for Event LY038 DNA are included in Table 2.

One skilled in the art would include the appropriate control sampleswhen performing the PCR or Taqman® assays described in the presentinvention. The inclusion of positive control DNA samples, negativecontrol DNA samples, and other controls are appropriate and aid in theinterpretation of results. In addition, the person of ordinary skill inthe art would know how to prepare internal control primers and probesfor the Taqman® PCR reaction using published, standard methods (aspublished by, for example, Applied Biosystems, Foster City, Calif.). Oneskilled would also realize that the particular primer sequences, probes,and reaction conditions specified herein may be modified and produce anassay which is diagnostic for Event LY038 DNA. Additionally, one skilledin the art would know that the products of the PCR reaction may beanalyzed by gel electrophoresis for analysis.

TABLE 2 PCR reaction mixture and conditions diagnostic for LY038 eventDNA Step Reagent Volume Comments 1 18 megohm water Adjust for e.g. SigmaCatalog #W-4502 final volume of 10 μl 2 2 × Universal Master Mix   5 μlApplied Biosystems, Palo Alto, CA; Part # 4304437; 1 × finalconcentration of buffer 3 Mixture of event-specific 0.5 μl  1 μM finalconcentration primers (SEQ ID NOs: 3 and 4)* 4 Event-specific probe FAMlabel 0.2 μl 200 μM final concentration (SEQ ID NO: 5)** 5 Mixture ofinternal control 0.5 μl  1 μM final concentration primers*,{circumflexover ( )} 6 Internal control probe VIC label 0.2 μl 200 μM finalconcentration 7 DNA template*** 3.0 μl Preferably 5–10 ng per reaction 8Amplification Cycle No. Settings 1   50° C. 2 minutes 1   95° C. 10minutes 10   95° C. 15 seconds   64° C. 1 minute  −1° C./cycle 30   95°C. 15 seconds   54° C. 1 minute 1   10° C. forever 9 Analysis of PCRreaction Applied Biosystems Gene|Amp PCR System 9700 or fluorescentplate reader *Resuspend mixed primers in 18 megohm water to aconcentration of 20 μM each primer. Example: 100 μl first primer at aconcentration of 100 μM, 100 μl second primer at a concentration of 100μM, 300 μl 18 megohm water. **Resuspend probe in 18 megohm water to aconcentration of 10 μM. ***May include but not be limited to: negativeDNA control (e.g., non-transgenic DNA) negative water control (notemplate DNA) positive control (Event LY038) sample DNA (from leaf,seed, other plant part samples) {circumflex over ( )}Internal controlprimer and probe combinations may be made to a wide variety of genes orgenomic regions, the design of which is known to those of skill in theart.

Deposits of the Monsanto Company maize seed representative of EventLY038 disclosed above have been made under the Budapest Treaty with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110, on Oct. 29, 2003. The ATCC accession number forEvent LY038 is PTA-5623. The deposit will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that thepresent invention can be modified in arrangement and detail withoutdeparting from such principles. We claim all modifications that arewithin the spirit and scope of the appended claims.

1. A DNA construct comprising SEQ ID NO:7.
 2. The DNA construct of claim1, further comprising recombination sites.
 3. The DNA construct of claim2, wherein said recombination sites are lox P sites.
 4. A stablytransformed monocot plant comprising the DNA construct of claim 1 in itsgenome.
 5. The stably transformed monocot plant of claim 4, wherein saidplant is a variety of maize.
 6. The stably transformed monocot plant ofclaim 4, wherein the plant generates more lysine as compared to a secondplant of the same species that does not include the DNA construct. 7.The stably transformed monocot plant of claim 5, wherein said plant is aprogeny of a plant grown from seeds of ATCC seed deposit PTA-5623. 8.Progeny seed from the stably transformed maize plant of claim 5, whereinsaid seed comprises SEQ ID NO:7.
 9. A method of producing a progenymaize plant comprising Event LY038 comprising the step of crossing afirst parent maize plant regenerated from a seed wherein representativeseed having been deposited as ATCC Accession No. PTA-5623 and comprisingexogenous DNA insertion Event LY038, with a second parent maize plant,wherein said progeny plant comprises exogenous DNA insertion EventLY038.
 10. The method of claim 9, further comprising the steps of: (a)crossing the first progeny plant with itself or with a third maize plantto produce a seed of a second progeny plant of a subsequent generation;(b) growing a second progeny plant of a subsequent generation from saidseed and crossing the second progeny plant of a subsequent generationwith itself or a fourth maize plant to produce a seed of a third progenyplant of a subsequent generation; and (c) repeating steps (a) and (b)for at least one additional generation to produce an inbred maize plantcomprising exogenous DNA insertion Event LY038.
 11. The method of claim9, wherein said second maize plant does not comprise an exogenous DNAidentified as Event LY038 and wherein said second maize plant is of adifferent genotype than said maize plant comprising an exogenous DNAidentified as Event LY038, thereby producing a hybrid maize plant.
 12. Amethod of producing a maize seed having a higher lysine content,comprising (a) planting maize seed having in its genome a DNA moleculecomprising the DNA construct of claim 1; (b) growing plants from saidmaize seed, whereby said DNA molecule is expressed in maize cells toproduce maize seed having a higher lysine content than maize seedlacking said DNA molecule; and (c) harvesting said seed.
 13. The methodof claim 12, wherein said maize seed is from ATCC seed deposit PTA-5623or progeny thereof that comprise exogenous DNA insertion Event LY038.14. A maize meal comprising the construct of claim
 1. 15. The maize mealof claim 14, wherein said maize meal comprises tissue from a plant thatis crown from ATCC seed deposit PTA-5623 or progeny thereof, and whereinsaid meal comprises exogenous DNA insertion Event LY038, which isidentifiable by a DNA amplification method, wherein said method producesan amplicon comprising SEQ ID NO:1, 2, 5, 6, or
 11. 16. A maizeendosperm with increased lysine relative to non-transgenic maizeendosperms, wherein the maize endosperm comprises SEQ ID NO:5 and SEQ IDNO:11 and is derived from progeny of ATCC seed deposit PTA-5623, andwherein said endosperm is identifiable by a DNA amplification method,wherein said method produces an amplicon comprising SEQ ID NO: 1, 2, 5,6, or
 11. 17. A transgenic maize seed, wherein said seed is ATCC seeddeposit Accession No. PTA-5623 or progeny thereof, and wherein said seedcomprises SEQ ID NO:5 and SEQ ID NO:11 and wherein said seed hasincreased lysine levels relative to non-transgenic maize seeds.
 18. Amaize plant or parts thereof comprising SEQ ID NO:5 and SEQ ID NO:11,wherein said plant or parts thereof has increased lysine levels relativeto non-transgenic maize plants or parts thereof, and wherein said plantor Parts there of are produced by growing the seed of claim
 17. 19. Themaize plant or parts thereof of claim 18, wherein said parts thereof arepollen, ovule, seed, roots, or leaves.
 20. The maize plant of claim 19or progeny thereof wherein the genome of said maize plant or progenythereof comprises SEQ ID NO: 1, 2, 5, 6, and 11, and wherein saidprogeny thereof have increased lysine levels relative to non-transgenicmaize plants.
 21. A maize plant produced by growing seed from ATCC seeddeposit PTA-5623, or progeny of said plant, wherein said progeny haveincreased lysine levels relative to non-transgenic maize plants, andwherein the genome of said plant or progeny thereof comprises SEQ IDNO:1, 2, 5, 6, and
 11. 22. The maize plant or parts thereof of claim 18,wherein said plant or parts thereof comprises exogenous DNA insertionEvent LY038.
 23. The maize plant of claim 21, wherein said plant isidentifiable by a DNA amplification method wherein said method producesan amplicon comprising SEQ ID NO:1, 2, 5, 6, or
 11. 24. A maize planthaving increased lysine relative to non-transgenic maize plantscomprising incorporated in its genome a Corynebacterium DHDPS encodingDNA molecule, and SEQ ID NO:5 and SEQ ID NO:11, wherein said maize plantis derived from progeny of ATCC seed deposit PTA-5623.
 25. The maizeseed of claim 17, wherein said seed comprises exogenous DNA insertionEvent LY038.
 26. The maize seed of claim 17, wherein said seed isidentifiable by a DNA amplification method, wherein said method producesan amplicon comprising SEQ ID NO:1, 2, 5, 6, or
 11. 27. The maizeendosperm of claim 16, wherein said endosperm comprises exogenous DNAinsertion Event LY038.
 28. A maize meal with increased lysine relativeto maize meals made from non-transgenic maize seeds, wherein the maizemeal is derived from progeny of ATCC seed deposit PTA-5623, and whereinsaid maize meal comprises SEQ ID NO:5 and SEQ ID NO:11.
 29. A method ofproducing a maize plant comprising Event LY038 comprising the steps of:(a) crossing a maize plant with another maize plant comprising exogenousDNA insertion Event LY038, wherein said maize plant comprising EventLY038 is grown from ATCC seed deposit PTA-5623 or is progeny thereof;(b) obtaining at least one progeny plant derived from the cross of (a);and (c) selecting progeny plants that comprise Event LY038.
 30. A methodof producing a maize plant with increased lysine relative tonon-transgenic maize plants comprising the steps of: (a) crossing amaize plant with another maize plant comprising the nucleotide sequencesof SEQ ID NO:5 and SEQ ID NO:11, wherein said maize plant comprising SEQID NO:5 and SEQ ID NO:11 is grown from ATCC seed deposit PTA-5623 or isprogeny thereof; (b) obtaining at least one progeny plant; and (c)selecting progeny plants comprising nucleotide sequences of SEQ ID NO:5and SEQ ID NO:11 and which have increased lysine compared tonon-transgenic maize plants.